Generally, to amplify DNA (Deoxyribose Nucleic Acid) using the PCR process, it is necessary to cycle a specially constituted liquid reaction mixture through several different temperature incubation periods. The reaction mixture is comprised of various components including the DNA to be amplified and at least two primers sufficiently complementary to the sample DNA to be able to create extension products of the DNA being amplified. A key to PCR is the concept of thermal cycling: alternating steps of melting DNA, annealing short primers to the resulting single strands, and extending those primers to make new copies of double-stranded DNA. In thermal cycling the PCR reaction mixture is repeatedly cycled from high temperatures of around 90° C. for melting the DNA, to lower temperatures of approximately 40° C. to 70° C. for primer annealing and extension. Generally, it is desirable to change the sample temperature to the next temperature in the cycle as rapidly as possible. The chemical reaction has an optimum temperature for each of its stages. Thus, less time spent at non optimum temperature means achieving better chemical results. Also a minimum time for holding the reaction mixture at each incubation temperature is required after each said incubation temperature is reached. These minimum incubation times establish the minimum time it takes to complete a cycle. As such, any transition time between sample incubation temperatures is time added to this minimum cycle time. Since the number of cycles is fairly large, this additional time unnecessarily heightens the total time needed to complete the amplification.
In some previous automated PCR instruments, sample tubes are inserted into sample wells on a thermal block assembly. To perform the PCR process, the temperature of the thermal block assembly is cycled according to prescribed temperatures and times specified by the user in a PCR protocol file. The cycling is controlled by a computing system and associated electronics. As the thermal block assembly changes temperature, the samples in the various tubes experience similar changes in temperature. However, in these previous instruments differences in sample temperature are generated by thermal non-uniformity (TNU) from place to place within the thermal block assembly. Temperature gradients exist within the material of the block, causing some samples to have different temperatures than others at particular times in the cycle. Because the chemical reaction of the mixture has an optimum temperature for each or its stages, achieving that actual temperature is critical for good analytical results. A large TNU can cause the yield of the PCR process to differ from sample vial to sample vial.
As such, the analysis of TNU is an important attribute for characterizing the performance of a thermal block assembly, which may be used in various bioanalysis instrumentation. The TNU is typically measured in a sample block portion of a thermal block assembly, and is typically expressed as either the difference or the average difference between the hottest well and the coolest position on the sample block portion engaging a sample or samples. The industry standard, set in comparison with gel data, a difference of about 1.0° C., or an average difference of 0.5° C. Historically, the focus on reducing TNU has been focused on the sample block. For example, it has been observed that the edges of the sample block are typically cooler than the center. One approach that has been taken to counteract such edge effects is to provide various perimeter and edge heaters around the sample block to offset the observed thermal gradient from the center to the edges.